Figure 1—source data 1

Intra-kinetochore distances with and without microtubule attachment.

Table of measurements of Δ 3D for all kinetochore markers imaged in untreated and 3 µM nocodazole-treated cells. Values given are medians ± standard error, and p values were given by Mann-Whitney U tests.

Intra-kinetochore distances with and without microtubule attachment.

Schematic of the 3D mammalian kinetochore with (top) and without (bottom) attachment to a kinetochore-microtubule (kMT). The difference between endogenous CENP-A and eGFP-CENP-A has been attributed to limitations of antibody accessibility to CENP-A that is deep in centromeric chromatin (Magidson et al., 2016). As previously found (Suzuki et al., 2015) the majority of CENP-C amino-termini are 'free' and in close proximity of the centromere, rather than directly bound to the 20 nm long MIS12 complex (Petrovic et al., 2014). The orientation of the NDC80 complex is suggested based on the 60 nm length of the complex and the flexibility in the hinge region (Wei et al., 2005; Wang et al., 2008). Bub3 binds to kinetochores through the phospho-MELT repeats in KNL1 suggesting that this protein extends beyond the NDC80 complex – at least during metaphase.

Chromatic shift correction precision and accuracy in the dynamic live assay to analyse intra-kinetochore measurements.

(A) Histograms representing the compilation of all measurements of x- (ζx; light blue), y- (ζy; purple), and z-directional (ζz; pink) chromatic shift, defined as the distance between the diffraction-limited spot centres of the two fluorophores, in cells expressing eGFP-CENP-A and mCherry-CENP-A, for 18 imaging sessions. Each distribution of measurements of chromatic shift for a given imaging session was shifted by its median (ζ¯x, i.e. the measured chromatic shift for that imaging session) to centre it at zero. Values given are means of the standard deviations for each of these distributions (n = 18). (B) Schematic demonstrating the expected orientation of a kinetochore’s inner (green circles) and outer domains (red circles) relative to its spindle pole (black crossed circles) in both the xy- (top) and xz-plane (bottom). Blue lines represent the k-fibres which bind the kinetochores and spindle poles. Thin dashed red circles represent examples of biological variance in outer domain localisation, thick red circles represent the expected outer domain localisation. Dark blue arrows represent measurements of x- (Δx), y- (Δy) and z-directional (Δz) intra-kinetochore distance. (C) Histograms of measurements of x- (Δx; light blue), y- (Δy; purple) and z-directional (Δz; pink) intra-kinetochore distance from dynamic live movies of cells expressing eGFP-CENP-A and Ndc80-tagRFP. Values given are medians ± standard error, which are close to zero as expected from the schematic in (B).

Figure 2—source data 1

Table of measurements of Δ 1D, Δ 2D, ϑ 3D-swivel and standard deviation of ϑ y-swivel distribution for all kinetochore markers imaged in untreated and 3 µM nocodazole-treated cells. n values correspond to all measurements in columns to the left of the n-value column. Values given are medians ± standard error.

Kinetochore swivel is also present between other pairs of kinetochore markers.

Example images of other pairs of kinetochore proteins tagged by fluorescent markers with visible swivel, including (clockwise from top left): CENP-A-Alexa488 and Ndc80-tagRFP; eGFP-CENP-A and Nnf1-Alexa594; Bub3-eGFP and mCherry-CENP-A; GFP-CENP-O and Ndc80-tagRFP; GFP-CENP-C and Ndc80-tagRFP; GFP-CENP-C and mCherry-Ndc80; CENP-C-Alexa488 and mCherry-Ndc80; and eGFP-CENP-A and mCherry-Mis12. Green dashed lines are the sister-sister axes, white dashed lines are intra-kinetochore axes. Scale bars 500 nm.

Outer kinetochore components are capable of ‘swivel’ about the inner kinetochore, which increases upon microtubule depolymerisation.

(A) Example images of kinetochore pairs in untreated (top) and 3 µM nocodazole (bottom) cells exhibiting swivel in each of its kinetochores. Green and white dashed lines represent sister-sister and intra-kinetochore axes, respectively. Associated schematic shows measurements of swivel (ϑ swivel, between the sister-sister and marker-marker axes), and twist (ϑ twist, between the sister-sister axis and the metaphase plate). Stated values given are ϑy-swivel at each kinetochore. Scale bars 500 nm. (B) Distributions of ϑ swivel in 3D (histograms in top row) and projected on the y-axis (rose plots in bottom row), in untreated (blue; n = 4291) cells compared to 3 µM nocodazole (gold; n = 649), each significantly different to untreated (p < 10–4). Values given are medians ± standard error in ϑ swivel, and standard deviation, σ, for ϑy-swivel. Statistical tests were Mann-Whitney U for ϑ swivel, and F test for ϑy-swivel. (C) A kinetochore pair in untreated cells exhibiting temporally-changing swivel. Time is given at the top right of each frame. Values given are ϑy-swivel at each kinetochore for each time point. Scale bar 500 nm.

Figure 4—source data 1

Swivel decrease is partnered by an increase in 1D at anaphase onset, but 3D is invariant.

Median and standard error values for the bar plots in Figure 4E of ϑy-swivel and 1D (left) and 3D (right) measurements of Δ, comparing kinetochore pairs during early and late metaphase, shown in Figure 4E. ϑy-swivel and Δ 1D significantly increase and decrease, respectively, however 3D does not change.

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